In winemaking, controlling the final acidity of the wine can be important, particularly to impart complexity of aroma and flavor and to increase biological stability of the wine. For example, red and white wines produced in cooler climatic regions frequently have excess acidity, principally because of elevated levels of malic acid in the wine. Such acidic wines usually require deacidification. Wines produced in warmer climates generally have lower acidity. Consequently, they often do not require deacidification.
Malolactic fermentation (MLF) is a metabolic process performed by certain lactic acid bacteria (LAB), which can be utilized to reduce wine acidity. In this process, malic acid (which has two carboxylic acid groups) is decarboxylated to form lactic acid (which has only one carboxylic acid group) and carbon dioxide. In the decarboxylation reaction, a proton (H.sup.+) becomes covalently bonded to the lactic acid. Since protons in the wine are thereby consumed, wine pH is correspondingly increased.
The LAB capable of performing MLF are principally from the genera Leuconostoc, Lactobacillus, and Pediococcus. Such bacteria exist naturally, along with other bacteria, on the surfaces of grape leaves and skins. Wibowo et al., "Occurrence and Growth of Lactic Acid Bacteria in Wine: A Review," Am. J. Enol. Vitic. 36:302-313 (1985). However, relying on indigenous populations of LAB comprising multiple species, some of which perform undesirable metabolic reactions on wine constituents, to perform MLF can be risky because many indigenous LAB species produce undesirable flavors and odors in wines. In addition, growth of indigenous LAB in wine can result in other forms of wine spoilage such as ropiness (formation of mucilaginous material) and formation of excess acetic acid. Suppression of LAB species can be achieved in some instances by maintaining a wine pH of less than about 3.3, an alcohol content greater than about 14% v/v, and a sulfur dioxide concentration greater than about 50 mg/L. However, these conditions may be neither desirable nor feasible for many wines.
As a result, many winemakers faced with the problem of acid reduction in a new wine inoculate the wine with a pure starter culture comprised of a suitable LAB, particularly Leuconostoc oenos. L oenos is the only known species of LAB capable of growing and degrading malic acid in low pH environments (less than about 3.5) and in the presence of ethyl alcohol. Henick-Kling et al., "Evaluation of Malolactic Bacteria Isolated from Oregon Wines," Appl. and Environm. Microbiol. 55:2010-2016 (1989); Watson, "Malolactic Fermentation," Wine Advisory Bd. Res. Rept., Issue 2, Oregon Dept. of Agric. (Jan. 1986). Using pure starter cultures has the advantage of not having to rely on the uncertain prospects of employing a mixed population of indigenous LAB to decarboxylate malic acid. However, use of pure starter cultures alone provides no assurance that undesirable LAB that may be present in the wine will not propagate and eventually spoil the wine, especially after MLF has progressed sufficiently to elevate the pH to a level permissive for such propagation.
L. oenos starter cultures are typically prepared by growing a population of the bacteria in an aqueous culture medium. A volume of the starter culture is then added to the wine, generally during or after the primary fermentation is complete. Although L. oenos can withstand a wine environment, after a starter culture thereof is added to the wine, certain strains of these bacteria comprising the inoculum can experience a substantial shock resulting from their being transferred from a relatively mild culture-medium environment to a wine environment. As a result, the bacteria can experience a partial decline in numbers, followed by a recovery of viable bacteria. After the LAB population density subsequently reaches about 10.sup.6 cells/mL, MLF becomes pronounced. See Wibowo et al., Am. J. Enol. Vitic. 36:302-313 (1985).
In view of the partial die-off of inoculum bacteria, many winemakers inoculate the wine with a starter culture comprising a large number of viable L. oenos bacteria in the hope that MLF will begin rapidly and the inoculum bacteria will be able to overwhelm the activity of any undesirable LAB in the wine. However, use of a large starter culture provides no guarantee that undesirable LAB will be sufficiently outnumbered so as to have a negligible detrimental effect on the wine.
Many winemakers attempt to inhibit LAB and other bacteria as well as spoilage yeast in wine by adding sulfur dioxide (SO.sub.2) thereto. This is a widespread practice. Unfortunately, sulfur dioxide also inhibits L. oenos and is a substance to which some people are sensitive or allergic. Since such people must avoid drinking wine containing sulfur dioxide, it is not surprising that many wine producers would like to reduce or eliminate use of sulfur dioxide. Nevertheless, these same winemakers have persisted in using sulfur dioxide because there has as yet been no feasible alternative for controlling undesirable LAB in a consistent and predictable manner, especially in wines that have been minimally processed.
Recent studies have shown that wine LAB can be inhibited by a bacteriocin termed "nisin." Radler, "Possible Use of Nisin in Winemaking. I. Action of Nisin Against Lactic Acid Bacteria and Wine Yeasts in Solid and Liquid Media," Am. J. Enol. Vitic. 41:1-6 (1990); Radler, "Possible Use of Nisin in Winemaking. II. Experiments to Control Lactic Acid Bacteria in the Production of Wine," Am. J. Enol. Vitic. 41:7-11.
Nisin is produced by Streptococcus lactis bacteria, which belong to the serological group N. The term "nisin" is derived from the phrase "N inhibitory substance." Hurst, "Nisin and Other Inhibitory Substances from Lactic Acid Bacteria," in Branell et al. (eds.) Antimicrobials in Foods, Dekker, pp. 327-351 (1983). Nisin is not active against gram-negative bacteria, fungi or yeasts. It is active against virtually all LAB. The mechanism of nisin action is presently not fully understood, although it appears to cause rupture of bacteria cell membranes.
The nisin polypeptide is generally regarded as a dimer having a molecular weight of about 7000 daltons and is comprised of 34 amino acid residues, some of which are rare in nature. Lipinska, "Nisin and Its Applications," in Woodbine (ed.) Antibiotics and Antibiosis in Agriculture, Butterworth, London, pp. 103-130 (1977).
Nisin has achieved widespread acceptance outside the United States as a preservative in foods, especially in dairy products and canned vegetables and meats. Hall, "Nisin and Food Preservation," Process Biochem. (Dec. 1966), pp. 461-464. In fact, nisin occurs naturally in certain foods, especially dairy products, that are made by a process that includes a fermentation step involving S. lactis. A recent report espouses the use of nisin in beer brewing, where the bacteriocin limits undesired LAB without affecting yeast fermentation or beer flavor. Ogden, "Nisin: A Bacteriocin with a Potential Use in Brewing," J. Inst. Brew. 92:379-383 (1986).
After 25 years of safe use in many European countries, nisin was recently affirmed by the Food and Drug Administration as Generally Recognized As Safe (GRAS) for use in inhibiting growth of Clostridium botulinum in pasteurized process cheese spreads. See 53 Fed. Reg. 1247-11251 (1988). This action was based on the large accumulated body of scientific data indicating that nisin is non-toxic, non-allergenic, and is a safe and effective antimicrobial agent.
Unfortunately, nisin is active against many bacteria capable of performing MLF in wine. For example, L. oenos will not grow at nisin concentrations greater than about 1 Unit/mL.
Nisin production is governed by at least one DNA sequence present in nisin-producing microorganisms such as S. lactis. The DNA sequence is apparently borne on a plasmid, which can be transferred to other microorganisms via gene cloning techniques known in the art. For example, U.S. Pat. No. 4,716,115 to Gonzalez et al. discloses the transfer of nisin-encoding DNA into recipient microorganisms that naturally lack the ability to produce the bacteriocin. The resulting "transformed" recipient microorganisms thereby become nisin-producing. Gonzalez et al. discloses that incorporation of nisin genes into nisin-resistant lactic acid bacteria can yield nisin-producing transformants usable for preserving foods such as meat and dairy products. However, Gonzalez et al. does not disclose how to generate nisin-resistant LAB capable of conducting MLF in wine.
Hence, there is a need for a method for reducing the acidity of wine by malolactic fermentation wherein the winemaker has complete control over when during a winemaking process a malolactic fermentation step occurs, particularly without the hazard of wine spoilage due to the unintentional propagation of harmful bacteria in the wine.
There is also a need for such a method in which malolactic fermentation of the wine can be performed by inoculating the wine with a "pure culture" of a suitable lactic acid bacteria without the need to pre-sterilize the wine in an effort to suppress the propagation of wine-spoilage bacteria.
There is also a need for such a method that does not detrimentally alter the taste or aroma of the wine.